Recombinant protein production is an essential activity for high throughput screening, functional validation, structural biology, and production of pharmaceutical polypeptides. Escherichia coli is a widely used organism for the expression of heterologous proteins because it easily grows to a high cell density on inexpensive substrates, and has well-established genetic techniques and expression vectors. However, this is not always sufficient for the efficient production of active biomolecules. In order to be biologically active, polypeptide chains have to fold into the correct native three-dimensional structure, including the appropriate formation of disulfide bonds, and may further require correct association of multiple chains.
Although the active state of the protein may be thermodynamically favored, the time-scale for folding can vary from milliseconds to days. Kinetic barriers are introduced, for example, by the need for alignment of subunits and sub-domains. And particularly with eukaryotic proteins, covalent reactions must take place for the correctly folded protein to form. The latter types of reaction include disulfide bond formation, cis/trans isomerization of the polypeptide chain around proline peptide bonds, preprotein processing and the ligation of prosthetic groups. These kinetic limitations can result in the accumulation of partially folded intermediates that contain exposed hydrophobic ‘sticky’ surfaces that promote self-association and the formation of aggregates.
The expression of multimeric proteins such as hormones and enzymes in active form in particular is difficult to achieve in some recombinant expression systems. Many host cells do not possess the appropriate enzymes to process such proteins in biologically active form. Because of these difficulties such multimeric proteins usually must be expressed in mammalian cells. This unfortunately raises the costs associated with producing the protein as well as resulting in issues of viral contamination which are particularly problematic if the multimeric protein is to be used a therapeutic. One prevalent example of a multimeric protein typical produced in mammalian culture systems such as CHO cells is recombinant immunoglobulins or antibodies.
Antibodies are tetrameric proteins, which have many uses in clinical diagnosis and therapy. Each antibody tetramer is composed of two identical light chains and two identical heavy chains. Pure human or humanized antibodies of a specific type are difficult or impossible to purify in sufficient amounts for many purposes from natural sources. As a consequence, biotechnology and pharmaceutical companies have turned to recombinant DNA-based methods to prepare them on a large scale. The production of functional antibodies requires not just the synthesis of the two polypeptides but also a number of post-translational modifications, including proteolytic processing of the N-terminal secretion signal sequence; proper folding and assembly of the polypeptides into tetramers; formation of disulfide bonds; and specific N-linked glycosylation. All of these events take place in the eukaryotic cell secretory pathway, an organelle complex unique to eukaryotic cells.
Recombinant synthesis of such complex proteins has had to rely on higher eukaryotic tissue culture-based systems for biologically active material. However, as mentioned mammalian tissue culture based production systems are significantly more expensive and complicated than microbial fermentation methods. In addition, there continues to be questions regarding therapeutic products produced using materials derived from animal by-products.
Alternatives to mammalian expression systems for the expression of recombinant proteins are eukaryotic microbia such as yeast and insect cell expression systems. Insect cell expression systems use baculovirus vectors and often achieve high yields of secreted proteins. However, such cells are not always capable of processing complex mammalian polypeptides in active form. Yeast find fairly well established usage in expressing recombinant proteins. The most typically used yeast is Saccharomyces since it has been well characterized, many promoters suitable for use therein are widely available as are sequences for facilitating secretion such as the alpha and A factor secretory signal sequences. Another yeast which has been suggested to be capable of producing mammalian proteins in active form is Pichia, and particularly Pichia pastoris. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression levels. As a yeast, it shares the advantages of molecular and genetic manipulations with Saccharomyces. These features make Pichia very useful as a protein expression system. In addition various other types of yeast have been disclosed to be suitable for expression of heterologous polypeptides.
Many of the techniques developed for Saccharomyces may be applied to Pichia as well as to other types of yeast. These include transformation by complementation; gene disruption and gene replacement. In addition, the genetic nomenclature used for Saccharomyces has been applied to Pichia. There is also cross-complementation between gene products in both Saccharomyces and Pichia. Several wild-type genes from Saccharomyces complement comparable mutant genes in Pichia. 
Heterologous expression in Pichia pastoris as well as other types of yeast can be either intracellular or secreted. Secretion requires the presence of a signal sequence on the expressed protein to target it to the secretory pathway. While several different secretion signal sequences have been used successfully, including the native secretion signal present on some heterologous proteins, success has been variable. A potential advantage to secretion of heterologous proteins is that Pichia pastoris secretes very low levels of native proteins. That, combined with the very low amount of protein in the minimal Pichia growth medium, means that the secreted heterologous protein comprises the vast majority of the total protein in the medium and serves as the first step in purification of the protein.
Many species of yeast, including Pichia, are mating competent. This enables two distinct haploid strains to mate naturally and generate a diploid species possessing two chromosomal copies Alternatively, polyploid yeast can be obtained by artificial methods, i.e., spheroplast fusion.
As noted yeast including Pichia have been used many years for the production of heterologous proteins. Although P. pastoris in particular has been used successfully for the production of various heterologous proteins, e.g., hepatitis B surface antigen (Cregg et al. (1987) Bio/Technology 5:479), lysozyme and invertase (Digan et al. (1988) Dev. Indust. Micro. 29:59; Tschopp et al. (1987) Bio/Technoloqy 5:1305), endeavors to produce other heterologous gene products in Pichia, especially by secretion, have given mixed results. At the present level of understanding of the P. pastoris expression system, it is unpredictable whether a given gene can be expressed to an appreciable level in this yeast or whether Pichia will tolerate the presence of the recombinant gene product in its cells. Further, it is especially difficult to foresee if a particular protein will be secreted by P. pastoris, and if it is, at what efficiency.
Additionally, prior to the present invention the use of diploid yeast to secrete heterologous polypeptides had not been reported. Rather, the earlier work using yeast to produce secreted heterologous polypeptides was limited to haploidal yeast expression systems. In fact, earlier evidence suggested that diploid yeast such as Pichia would be incapable of stably expressing and secreting heterologous polypeptides in amounts required for such expression systems to be suitable for commercial use.
The present invention therefore provides improved methods and compositions of matter that provide for the secretion of heterologous polypeptides, preferably heteromultimers using polyploidal yeast cultures preferably produced from mating competent yeast, including Pichia and other genera.